SUMMARY Phenotypic shifting and migration of vascular smooth muscle cells (VSMCs) play key roles in the progression of atherosclerosis. Phenotypic switching in VSMCs is accompanied by the differential expression of integrins, which is involved in cell-extracellular matrix (ECM) adhesion and VSMC migration. Substantial progress has been made to advance understanding of the mechanism of VSMC migration in atherosclerosis. Little, however, is known about how cholesterol directly affects the adhesive state and responsiveness of VSMCs to extracellular mechanical stimulation. This study investigates the molecular mechanism underlying the synergistic effect of cellular cholesterol and substrate stiffness on VSMC adhesion and migration. We will examine the expression and activity of integrin ?V?3, and N-cadherin (N-Cad), which have been reported to be involved in the development of atherosclerosis. Significantly, a more physiologically-relevant approach, statin, will be employed to manage cellular cholesterol instead of cyclodextrin following the guidelines of American Heart Association (AHA 2018). In a preliminary study, statin treatment reduced endogenous VSMC cholesterol without resulting in significant cell death compared to cyclodextrin. We also found that use of statin differentially affected integrins and N-Cad mediated VSMC adhesions. Based on these findings, we hypothesize that high cellular cholesterol and ECM stiffening synergistically induces the development of atherosclerosis by down-regulating N-Cad and integrin ?V?3-mediated cell adhesions, thereby enhancing VSMC migration for atherogenesis, while statin-mediated cholesterol depletion prevents the process by interfering with the biomechanics of VSMCs. This hypothesis will be tested by carrying out the following Specific Aims: (1) test the combined effects of statin-induced cholesterol depletion and substrate stiffening on the expression and activity of integrin ?V?3 and N-Cad in VSMCs; (2) test the combined effects of statin- induced cholesterol depletion and substrate stiffening on VSMC stiffness and cytoskeleton 3D architecture; and (3) test the combined effects of statin-induced cholesterol depletion and substrate stiffening on VSMC migration. The novelty of this work lies in linking VSMC mechanics and cytoskeletal organization with the development of atherosclerosis. Use of innovative approaches, integrated confocal microscopy and atomic force microscopy to monitor cell mechanics and cytoskeletal remodeling, as well as use of a state-of-the-art image processing technique, will enable an in-depth study of the contribution of VSMC mechanics and cytoskeleton architecture to atherosclerosis. This study will strengthen undergraduate student research activities in biomedical engineering at University of South Dakota by recruiting six undergraduate students for summer research (two for each year). Through their participation, students will gain substantial knowledge, skills, and experience in biomedical research, which will serve them well for their future career development.